Early Forebrain Wiring: Genetic Dissection Using
Conditional Celsr3 Mutant Mice
Libing Zhou, et al.
Science 320, 946 (2008);
DOI: 10.1126/science.1155244
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Early Forebrain Wiring: Genetic
Dissection Using Conditional
Celsr3 Mutant Mice
Libing Zhou,1 Isabelle Bar,2* Younès Achouri,1 Kenneth Campbell,3 Olivier De Backer,2
Jean M. Hebert,4 Kevin Jones,5 Nicoletta Kessaris,6 Catherine Lambert de Rouvroit,2
Dennis O’Leary,7 William D. Richardson,6 Andre M. Goffinet,1 Fadel Tissir1†
Development of axonal tracts requires interactions between growth cones and the environment.
Tracts such as the anterior commissure and internal capsule are defective in mice with null
mutation of Celsr3. We generated a conditional Celsr3 allele, allowing regional inactivation.
Inactivation in telencephalon, ventral forebrain, or cortex demonstrated essential roles for
Celsr3 in neurons that project axons to the anterior commissure and subcerebral targets, as well as
in cells that guide axons through the internal capsule. When Celsr3 was inactivated in cortex,
subcerebral projections failed to grow, yet corticothalamic axons developed normally, indicating
that besides guidepost cells, additional Celsr3-independent cues can assist their progression. These
observations provide in vivo evidence that Celsr3-mediated interactions between axons and
guidepost cells govern axonal tract formation in mammals.
ormation of axonal tracts is essential for
brain wiring, and several cues, such as
extracellular molecules, guidepost cells,
and fiber-to-fiber interactions, guide growing
axons to their targets (1). We showed previously that the anterior commissure (AC) and
the internal capsule (IC) are defective in constitutive Celsr3 mutant mice (2). Celsr1, Celsr2,
F
1
Developmental Neurobiology, Université Catholique de
Louvain, 1200 Bruxelles, Belgique. 2Facultés Universitaires
Notre-Dame de la Paix, 5000 Namur, Belgique. 3Division of
Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH 45229, USA. 4Albert Einstein College of
Medicine, Bronx, NY 10461, USA. 5University of Colorado,
Boulder, CO 80309, USA. 6University College London, London
WC1E 6AE, UK. 7Salk Institute, La Jolla, CA 92037, USA.
*Present address: Université Libre de Bruxelles, 1050 Bruxelles,
Belgique.
†To whom correspondence should be addressed. E-mail:
fadel.tissir@uclouvain.be
Celsr3 are homologous to Drosophila flamingo/
starry night (Fmi/stan) (3, 4), which collaborates
with Frizzled and Van Gogh to regulate planar
cell polarity (PCP) and neurite development.
Celsr proteins are seven-pass transmembrane
cadherins and are thought to mediate cell adhesion via homophilic interactions. Celsr3 is
expressed in postmigratory neurons in cortex,
ventral telencephalon, olfactory structures, and
thalamus during development and progressively down-regulated during maturation (5).
To probe forebrain wiring, we generated a
conditional mutant allele that allows inactivation
of Celsr3 by crosses with mice that express the
Cre recombinase in region-specific manners (6).
This allele was produced by flanking exons 19 to
27, deletion of which generates a null allele (2),
with loxP sites (“floxed” allele, Celsr3f ). Mice
with regional inactivation were produced by crossing double heterozygous males (Celsr3+/−;Cre/+)
with homozygous Celsr3f/f females. Celsr3 inactivation requires Cre-mediated modification of
one floxed allele only, thereby increasing efficiency. To facilitate reading, we use the shorthand
“|”: for example, Celsr3|Foxg1 is short for
Celsr3f/–;Foxg1-Cre/+. Cre-expressing strains
were crossed with ROSA26R mice (7) to verify
that Cre activation proceeded as described. Inactivation was further confirmed by in situ hybridization with a probe complementary to exons
19 to 27 that allows detection of intact Celsr3
mRNA only and by Western blot (fig. S1). Control animals were double heterozygotes, e.g.,
Celsr3f/+;Foxg1-Cre/+.
We first examined the role of Celsr3 during
formation of the AC. In Celsr3|Foxg1 mice in
which Celsr3 is inactivated in the telencephalon
(8), the AC was absent (Fig. 1, A, B, D, and E).
It was also drastically affected in Celsr3|Emx1
mice that express Cre in olfactory structures
and neocortex (9). Diminutive bundles originating from olfactory nuclei ran caudally and
turned, but never crossed the midline, as confirmed by injecting 1,1´-dioctadecyl-3,3,3´,3´tetramethylindocarbocyanine perchlorate (DiI)
in the olfactory bulb (Fig. 1, C and F). This
phenotype could reflect the kinetics of Cre activation: Axonal growth from olfactory nuclei
may be initiated before a full inactivation of
Celsr3 is achieved. In Celsr3|Emx1 mice, Celsr3
mRNA is absent from the olfactory and the
temporal neurons of origin of the AC but
present in the cells located along the pathway.
Thus, normal Celsr3 activity is likely required
cell-autonomously in the neurons of origin of
the AC. In all other crosses, namely Celsr3|Gsh2,
Celsr3|Nkx2.1, double Celsr3|(Gsh2 and Nkx2.1),
and Celsr|Dlx5/6, that express Cre in large
sectors of basal telencephalon but not in olfactory nuclei nor temporal cortex (10, 11), the AC
developed normally. This suggests that Celsr3
expression may not be required in cells along
the AC pathway. Alternatively, functionally
Fig. 1. Celsr3 expression is required
intrinsically in neurons of origin of the
AC. (A) Schematic representation of
the AC, composed of a rostral component (R) that contains commissural
axons connecting olfactory nuclei and
a caudal component (C) made of axons
that connect temporal lobes. OB indicates olfactory bulb. (B and C) Montages of coronal sections at the level of
the AC in newborn animals. The control phenotype is shown on the left
side, and the right side shows Celsr3|
Foxg1 (B) and Celsr3|Emx1 (C) mice.
Note the rudiment of AC in Celsr3|
Emx1 mice (arrow in C). (D to F) Horizontal sections at birth day (P0) after
DiI injection in control (D), Celsr3|
Foxg1 (E), and Celsr3|Emx1 mice (F).
Arrows in (C) and (F) point to rudiments of AC that never cross the midline.
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Fig. 2. Region-specific
Celsr3 inactivation affects development of
the IC in different manners. (A to F) Montages
of P0 sections stained
with Cresyl violet [(A) to
(C)] or neurofilament antibody (NF) [(D) to (F)].
The IC is fully defective
in Celsr3|Foxg1 mice
in which some thalamic
axons cross to the contralateral diencephalon
[arrow in (D)]. In Celsr3|
Dlx5/6 mice, the IC does
not form, but cortical
axons stall and form a
mass at the level of the
striatum [asterisks in (B)
and (E)], whereas thalamic fibers are misrouted to the amygdala
[arrow in (E)]. In Celsr3|
Emx1 mice, the IC is
present and thalamocortical connections are
similar to that in normal
mice [(C) and (F)]. (G)
Schematic summary of
the IC phenotypes in the
various mice used, in relation to areas of Celsr3
inactivation (gray) and
expression of markers
(Dlx5/6, Gsh2, Nkx2.1,
and Rora). dTh and vTh,
dorsal and ventral thalamus; HT, hypothalamus; VP, ventral pallidum; NCx, neocortex; PSPB, pallial subpallial boundary; and DTB, diencephalon telencephalon border.
Fig. 3. The IC corridor expresses Celsr3,
Dlx5/6, and Islet1 and is flanked with
expression of Nkx2.1 and Gsh2. Coronal sections at rostral (A to E) and
caudal levels (F to J) of the forebrain
at E12.5, showing expression of Celsr3
[(A) and (F), in situ hybridization], activation of the LacZ reporter in ROSA26R|
Nkx2.1 mice [(B) and (G)] and ROSA26R|
Gsh2 mice [(C) and (H)], expression of
Dlx5/6 [(D) and (I), EGFP in the transgene], and expression of Islet1 [(E) and
(J), immunohistochemistry]. A central
corridor is characterized by high levels
of Celsr3, Dlx5/6, and Islet1, and low
levels of LacZ. Arrows in (F) and (I)
indicate a Celsr3-positive, Dlx5/6negative zone that could explain partial growth of TCA in Celsr3|Dlx5/6
mice. CGE, LGE, and MGE, caudal, lateral, and medial ganglionic eminences.
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relevant cells in intermediate regions may have escaped Celsr3 inactivation, because Gsh2-, Nkx2.1-,
and Dlx5/6-Cre mice do not express Cre strongly in
the medial region where AC axons cross the midline. We favor the latter interpretation because it fits
with the observations on the IC described below.
We next investigated the function of Celsr3
during development of the three components of
the IC, namely corticothalamic axons (CTA), thalamocortical axons (TCA), and subcerebral projections [terminology of Molyneaux et al. (12)].
In Celsr3|Foxg1 mice, despite normal Celsr3 expression in dorsal thalamus, all three components
were defective (Fig. 2, A and D). No thalamic
fibers turned toward the striatum, and no thalamic
neurons were labeled after DiI injection in cortex.
Reciprocally, injection of DiI in thalamus did not
label cortical neurons but stained thalamic axons
that ran into the basal forebrain or crossed the
midline ventrally, like in constitutive Celsr3 and
Fzd3 mutants (2, 13) (fig. S2). With use of focal
DiI injections in the basal forebrain at embryonic day 13.5 (E13.5), we found that early
thalamic fibers reached the medial ganglionic
eminence in normal but not in Celsr3−/− mice.
In contrast, in both genotypes, corticofugal fibers crossed the pallial subpallial boundary and
reached the lateral ganglionic eminence at E13.5
(fig. S3). Thus, Celsr3 is required for the early
growth of TCA, but not CTA, toward ventral
telencephalon.
The three components of the IC were also
defective in Celsr3|Dlx5/6 mice that express Cre
in the ventral forebrain (11) (Fig. 2, B and E). A
subset of TCA managed an incomplete turning at
the diencephalon-telencephalon border but then
ran aberrantly through the pallidum and amygdala. Corticofugal fibers crossed the pallialsubpallial boundary and entered the lateral part of
the basal forebrain. However, they failed to progress and spiraled in a disorderly manner, forming an abnormal mass that filled most of the
dorsal striatum and protruded in the lateral ventricle (Fig. 2E). After DiI injections in cortex and
thalamus, no labeling of thalamic or cortical cells
was observed, confirming that both TCA and
CTA were defective (fig. S2). Similarly, no subcerebral projections formed, as shown by absence of corticospinal tract (CST) (fig. S4). Thus,
Celsr3 expression by Dlx5/6-positive cells is
required for progression of TCA, CTA, and
subcerebral projections through the ventral telencephalon. Partial progression of CTA through
the pallial subpial boundary in Celsr3|Dlx5/6 but
not in Celsr3|Foxg1 mice may reflect Celsr3mediated fiber-to-fiber interactions among CTA,
in which Celsr3 is expressed, or interactions between CTA and Celsr3-positive, Dlx5/6-negative
cells. Similarly, the partial turning of TCA in
Celsr3|Dlx5/6 mice may reflect interactions
with Celsr3-positive, Dlx5/6-negative cells.
To define which subset of Dlx5/6-positive
cells could qualify as guidepost cells, we used
Gsh2-Cre and Nkx2.1-Cre mice that express Cre
in the lateral and medial ganglionic eminences,
respectively. In Celsr3|Gsh2, Celsr3|Nkx2.1, and
double Celsr3|Gsh2-Nkx2.1 mice, all components of the IC developed normally (fig. S5).
This showed that a sufficient number of Celsr3expressing guidepost cells along the IC originate
Fig. 4. The corticospinal tract is defective in
Celsr3|Emx1 mice. Comparison of control [(A),
(C), and (E)] and Celsr3|
Emx1 [(B), (D), and (F)]
mice. In sagittal sections
at P6 stained with an
antibody against the L1
molecule (A) and (B), corticospinal axons are labeled in control [arrow
in (A)] but not in the
mutant ventral hindbrain.
Crosses were carried out
with Thy1-YFP mice, a
transgene that labels
neurons in cortical layer
5 and corticospinal axons
(C) to (F). At P20, layer 5
is well populated in control mice (C), and the
corticospinal tract is
clearly defined [arrows
in (E)], whereas cortical
layer 5 is very diminutive (D) and no corticospinal axons are detected
in the hindbrain (F) of
Celsr3|Emx1 mice.
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from Nkx2.1- and Gsh2-negative precursors. We
compared the distribution of Celsr3 (in situ hybridization), Nkx2.1 and Gsh2 (LacZ histochemistry and immunohistochemistry), Dlx5/6
[enhanced green fluorescent protein (EGFP) reporter], and Islet1 (immunohistochemistry) at
E12.5, before any fiber growth in the ventral
telencephalon, and at E14.5, when the IC begins to form. At E12.5 (Fig. 3), Celsr3 expression was very similar to that of Dlx5/6, with
maximal signal along the ganglionic eminences
where Islet1 was also present (compare Fig. 3, D
and E, to A). Expression of LacZ in ROSA26R|
Nkx2.1 and ROSA26R|Gsh2 mice was strong in
large sectors of the ganglionic eminences and in
the striatal mantle but low in the intermediate
region where expression of Celsr3 and Dlx5/6
was maximal. At E14.5 (fig. S6), zones of Gsh2
and Nkx2.1 expression flanked axonal bundles of
the incipient IC, whereas Islet1-expressing cells
were in close contact with axonal tracts, in the
region of highest Dlx5/6 signal and minimal Gsh2
and Nkx2.1 signal. These observations suggest
that Celsr3 is required in basal forebrain guidepost cells that are positive for Dlx5/6 and possibly Islet1 and that are derived from Nkx2.1-Cre–
and Gsh2-Cre–negative precursors.
To test whether Celsr3 is required intrinsically
for progression of corticofugal axons, we studied
Celsr3|Emx1 mice, in which Celsr3 is inactivated
early in the cortical anlage (9) (fig. S1). In those
mice, subcerebral projections such as CST were
defective. In crosses with Thy1–yellow fluorescent protein (YFP) transgenic mice (14), the CST
was clearly defined in control mice but absent
in Celsr3|Emx1 mice in which the number of cortical layer 5 neurons was dramatically reduced
(Fig. 4). After injections of Fluoro-Gold (Biotum,
Incorporated, Haywood, CA) in the spinal cord,
cells were labeled in the hindbrain, red nucleus,
and layer 5 in normal mice but only in the hindbrain and red nucleus, and not in layer 5, in
Celsr3|Emx1 mice (fig. S7). Thus, Celsr3 is required, presumably cell autonomously, in the
neurons of origin of subcerebral axons, like in
those of the AC.
In contrast to subcerebral projections, CTA
and TCA developed normally in Celsr3|Emx1
mice (Fig. 2). At E14.5, fibers from dorsal thalamus turned at the diencephalon-telencephalon
border and progressed along the ganglionic eminences before passing the pallial subpallial boundary and growing toward the cortex, similar to
fibers of control mice. Injections of DiI in the
cortex and thalamus resulted in labeling of thalamic and cortical neurons, respectively (fig. S2).
To assess the cortical distribution of TCA, we
studied cortical barrels by using cytochrome oxidase and Nissl staining of parietal cortex. As
shown in fig. S8, barrels failed to form in mice
that had no TCA, such as Celsr3|Foxg1 and
Celsr3|Dlx5/6 mice, and developed normally in
Celsr3|Emx1 mice, indicating normal TCA mapping. Thus, inactivation of Celsr3 in CTA did not
prevent them from navigating to the thalamus,
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an important developmental event that hitherto
received little attention.
In Drosophila wing cells, symmetrically expressed Fmi/stan proteins are thought to undergo
homophilic interactions, bringing distal and proximal cell membranes in contact and thereby fostering signaling by asymmetrically located Frizzled
on the distal and Van Gogh on the proximal side
(25). Axonal anomalies in Celsr3 and Fzd3 mutant mice are similar (13), suggesting that corresponding proteins also act together in mice (25).
Moreover, Fzd3 and Vangl2 are co-expressed with
Celsr3 in postmigratory neurons (26). Like in the
fly, Celsr3 expressed on the membranes of growth
cones and guidepost cells may promote adhesion
and allow Fzd3 and Vangl2 to interact and signal.
This model predicts that the expression and action
of Fzd3 and Vangl2 should be asymmetric, one in
axons and the other in guidepost cells. Conditional Fzd3 and Vangl2 alleles should allow testing that model further.
References and Notes
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27. We thank V. Bonte, I. Lambermont, and E. Paitre for
technical assistance; G. Hamard for help with
embryonic stem cell injections; M. Okabe for GFP mice;
F. G. Rathjen for antibodies against L1; P. Soriano for
ROSA26R mice; and L. Nguyen, A. Stoykova, and
P. Vanderhaeghen for help or discussion. This work was
supported by grants Actions de recherches concertées
(ARC-186), FRFC 2.4504.01, FRSM 3.4501.07, and FRSM
3.4529.03; by Interuniversity Poles of Attraction (SSTC,
PAI p6/20); and by the Fondation Médicale Reine
Elisabeth, all from Belgium. F.T. is a Research Associate
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nor did it perturb the growth and cortical mapping of TCA.
Why would Celsr3 be required in AC and
subcerebral axons but not in CTA? First, a few
subplate cells could escape Celsr3 inactivation in
Celsr3|Emx1 mice and provide pioneering axons
to thalamus (15, 16). However, Celsr3 is inactivated early in the cortex in those mice (fig. S1),
making this rather unlikely. Second, other Celsr
proteins may act redundantly with Celsr3 in CTA
neurons and mediate their interactions with
Celsr3-positive guidepost cells. Alternatively,
normal Celsr3 expression in dorsal thalamus
and basal forebrain in Celsr3|Emx1 mice allows
progression of TCA, which could encounter
Celsr3-deficient CTA and help them travel to
the thalamus, as predicted by the “handshake
hypothesis” (17). Celsr3|Rora mice were produced to inactivate Celsr3 in dorsal thalamic
nuclei and thereby assess their role in TCA
growth. The IC developed normally in those
mice, indicating a situation reciprocal to that in
Celsr3|Emx1 mice. However, studies of ROSA26R|
Rora mice showed that Cre expression was
restricted to a subset of dorsal thalamic cells.
Thus being unable to test the function of Celsr3
in thalamic neurons in vivo, we addressed the
question using explant cultures. We co-cultured
explants from normal or Celsr3-mutant thalamus
that expressed the GFP transgene ubiquitously
(18) with explants of normal ventral diencephalons at E13.5. As shown in fig. S9, normal dorsal thalamic axons were repelled by explants
from ventral diencephalon (32/57 cases) (19).
However, almost no repulsive activity was detected for Celsr3-defective thalamic axons (4/34
cases; P < 0.01, c2), suggesting that Celsr3 expression in TCA was required for their response
to ventral diencephalic cues. Thus, Celsr3 expression is probably necessary both in TCA and
in cells along their pathway (Celsr3|Dlx5/6 mice).
Our results have implications for the mechanisms of brain wiring and the function of Celsr3.
Demonstrated in invertebrates (20), a role of
guidepost cells in axonal navigation in mammals
has been repeatedly proposed (21–24). Our results demonstrate that they indeed play a crucial
function that requires Celsr3 expression (the role
of other molecules implicated in thalamocortical
and CST fiber nagivation is discussed briefly in
SOM). Altogether, our data indicate that Celsr3 is
required both in axons and guidepost cells, consistent with its mediating homophilic interactions.
Normal CTA development in Celsr3|Emx1 mice
indicates that Celsr3-independent cues are also
involved in their growth. Candidate mechanisms
are CTA-TCA fiber interactions like the handshake (17, 21), fiber-fiber interactions between
CTA and pioneer subplate axons (15, 16), and
adhesion of Celsr3 in guidepost cells with other
Celsr molecules present in growth cones. Furthermore, Celsr3|Emx1 mice provide a unique
model to study how subcerebral projections segregate from CTA when they reach the medial aspect of the IC en route to the cerebral peduncles,
Supporting Online Material
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Materials and Methods
SOM Text
Figs. S1 to S9
Table S1
References
15 January 2008; accepted 18 April 2008
10.1126/science.1155244
cAMP-Dependent Signaling as a Core
Component of the Mammalian
Circadian Pacemaker
John S. O’Neill,1* Elizabeth S. Maywood,1 Johanna E. Chesham,1
Joseph S. Takahashi,2 Michael H. Hastings1†
The mammalian circadian clockwork is modeled as transcriptional and posttranslational feedback
loops, whereby circadian genes are periodically suppressed by their protein products. We show
that adenosine 3′,5′-monophosphate (cAMP) signaling constitutes an additional, bona fide
component of the oscillatory network. cAMP signaling is rhythmic and sustains the transcriptional
loop of the suprachiasmatic nucleus, determining canonical pacemaker properties of amplitude,
phase, and period. This role is general and is evident in peripheral mammalian tissues and cell
lines, which reveals an unanticipated point of circadian regulation in mammals qualitatively
different from the existing transcriptional feedback model. We propose that daily activation of
cAMP signaling, driven by the transcriptional oscillator, in turn sustains progression of
transcriptional rhythms. In this way, clock output constitutes an input to subsequent cycles.
he suprachiasmatic nuclei (SCN) of the
hypothalamus are the principal circadian
pacemaker in mammals, driving the sleepwake cycle and coordinating subordinate clocks
T
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in other tissues (1). Disturbed circadian timing
can have a major negative impact on human
health (2). The molecular clockwork within the
SCN has been modeled as a combination of
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